Factores que afectan la compactación

Principle: HbF is not uniformly distributed among red cells, except in the condition of deletional HPFH. Cells with detectable amounts of HbF are called F-cells and they can be detected on blood smears by two techniques: the acid elution test of Kleihauer (13,14), and the immunofluorescence test using specific anti-HbF monoclonal antibodies (15,16). Methods of staining red cells in suspen- sion have been developed making possible the quantitation of erythrocytes by flow cytometry (17). Quantitative evaluation of F-cells may be useful to screen for HPFH, to monitor F cells in patients with sickle cell anaemia treated with hydroxyurea and to detect fetal cells in adult blood such as in case of fetal-maternal haemorrhage. The performance of different methods of flow cytometry for F cell counting has been recently reported (18).

2.8.1 F-CELLS BY ACID ELUTION TECHNIQUE

Principle: The acid elution technique is based on the differential elution of fetal and adult haemo-

1.

2. 3. 4. 5.

Reagents:

Citric-acid phosphate buffer pH 3.3:

(i) Solution A: (citric acid 0.1 M): 21.01g of citric acid in 1 litre of distilled water.

(ii) Solution B: (NaH₂PO₄ 0.2 M): 35.6 g NaH₂PO₄.2H₂O in 1 litre of distilled water.

Mix 73.4 ml of solution A with 26.6 ml of solution B; check pH and adjust if necessary to pH 3.3. Ethanol: 80 % vol.

Stains:

(i) 0.1% of erythrosine in water.

(ii) Erlich’s acid haematoxyline: dissolve 4.0 g of crystalline haematoxyline in 200 ml of ethanol 95% vol and add 8 ml of 10% sodium iodate. Add 200 ml of water and boil the mixture. Cool and add 200 ml of glycerin 6.0 g of aluminium ammonium sulphate and 200 ml of glacial acetic acid. Allow to stand the solution for at least 14 days.

Note: Complete kits of reagents are commercially available. Method:

Blood in any anticoagulant. Make thin smears and air dry for 10 to 60 minutes. Fix in ethanol 80 vol% for 5 minutes at 20-22°C.

Rinse the smears with tap water and air dry.

Stain with (i) for 3 minutes, then rinse with water and air dry. Counter-stain with (ii) for 3 minutes.

Rinse with tap water and air dry.

Examine under light microscopy without oil immersion.

Interpretation: F cells are densely stained with erythrosine. Cells containing HbA appear as ghost cells. Normal values for adults are below 0.01%.

Comments: The method is not quite sensitive and gives too low values. HbF at low concentration is eluted from red cells together with HbA. Pink stained cells are of difficult interpretation.

2.8.2 F-CELLS BY IMMUNOFLUORESCENCE

Materials:

Cleaned microscope slides: use pre-washed and pre-cleaned slides from BDH Superfrost (Cat.no. 406/0169/02).

(i) Clean slides with acetone followed by ethanol, and allow to air dry for at least 1 hour.

(ii) Prepare 4-5 very thin smears, one cell thick, per individual using 1 μl of fresh blood on the cleaned slides. Allow to dry for at least two days, for best results dry over one week.

Fixative: acetone/ethanol/methanol (6:2:2 v/v). Phosphate buffered saline (PBS) Sigma P-4417.

Trypsin solution: trypsin 0.1% in calcium chloride (CaCl2) 0.1% pH 7.8.

(i) Prepare 10 ml solution [10 mg of trypsin (ICN Cat.No.150213) +10 mg CaCl₂ +10 ml distilled wa- ter] and store at –20°C in 1 ml aliquots.

(ii) Alternatively, use trypsin tablets (Sigma; Cat.no. T-7168). Dissolve 1 tablet in 1 ml deionised

a. b. c. 1. 2. 3. 4. 5. 6. 7. a. b. c. d.

water, store at –20°C in 10 μl aliquots. Before use, make up to 500 μl (i.e. add 490 μl) with deionised water. Pre-warm to 37°C before use.

Note: The pH of CaC12 is very important.

Anti-γ monoclonal antibody (Sigma T-6653): undiluted supernatant.

Tetramethylrodamine Isothiocyanate (TRITC) conjugate anti-mouse IgG (Sigma T-6653).

(i) Store frozen in 10 μl aliquots.

(ii) Working solution: dilute 1:32 in PBS.

Humidity chamber: moist tissue in plastic chamber.

Glycerol: PBS (1:1 v/v) or anti-fade (Vectashield, Mounting Medium for Fluorescence; Vector Cat. No. H-1000).

Method:

Mark on the smear a small area of approximately 5 mm diameter using a diamond cutter.

Fix smears in acetone: ethanol: methanol fixative for 20 minutes. Air dry for 2 minutes (do not overdry).

Re-hydrate in PBS for 5 minutes (use plenty of PBS in a large glass container) and then rinse very briefly in distilled water. Air dry.

Cover the circled area with 8 μl of pre-warmed (37°C) trypsin solution and incubate at 37°C for 15 minutes in a humidity chamber.

Note: time of trypsinization varies with age of slides but generally 15 minutes is sufficient. Wash in PBS for 5 minutes with gentle agitation and rinse in distilled water. Air dry.

Cover trypsinized area with 5-10 μl anti-γ antibody, incubate at 37°C in humidity chamber for 30-40 minutes.

Wash as before and air dry.

Cover circled area with 5-10 μl fluorescent anti-mouse IgG and incubate at 37°C for 20-30 minutes. In the humidity chamber.

Wash as before with PBS, rinse in distilled water, air dry. Mount in Glycerol: PBS or anti-fade.

Interpretation and comments: We suggest - 1,000 red cells are counted, which corresponds to 4-5 high power fields. Adult normal values are 0.3 to 4.4% F-cells. Females have a higher number of F- cells than males. The number of fields, of course, depends on the density of cells. Hence, it is very important to master the art of making thin blood smears. Whole blood can be stored up to a week prior to making smears, but it is preferable to use fresh blood. The air-dried smears can be stored wrapped in tissue paper at room temperature for years.

2.9 HbA₂ DETERMINATION

This technique is discussed in chapter 3.

e. f. g. h. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.